Urokinase-type plasminogen activators (u-PAs) are found in at least three different forms in human urine, plasma and conditioned culture medium from a variety of cell lines. The first form to be characterized as u-PA consisted of a fibrinolytically active polypeptide of 410 amino acids, with an apparent moelcular weight of 54000 daltons, containing two disulfide-linked chains (Gunzler, W. A., Steffens, G. J., Oetting, F., Kim, SM. A. Frankus, E., and Flohe, L.: Hoppe--Seyler's Z. Physiol. Chem. 363, p. 1155, 1982).
The A-chain or light chain contains 157 amino acids and one triple disulfide-bonded "kringle" structure. This chain also contains a receptor binding domain for normal and neoplastic cells (monocytes, monocyte-like cells and A 431 epidermoid cells). The B chain or heavy chain (30000 daltons) consists of 253 amino acids and contains the catalytic domain.
This molecular form of u-PA is generally termed urokinase (UK), two chain urokinase (TC-UK) or high molecular weight urokinase (HMW-UK)(Gunzler, W. A., Steffens, G. J., Oetting, F., Buze, G., and Flohe, L.: Hoppe-Seyler's Z. Phisiol. Chem. 363, p. 133, 1982). The second form of u-PA has a molecular weight of 33000 daltons and results from proteolytic degradation of the HMW form by either plasmin or trypsin. It is called low molecular weight urokinase (LMW-UK). Protein sequence determinations have revealed that LMW-UK is identical to HMW-UK except for the absence of the NH2-terminal 135 amino acids that are specifically removed by the action of plasmin or trypsin (Steffens, G. J., Gunzler, W. A. Oetting, F., Frankus, E., and Flohe, L., Hoppe-Seyler's Z. Phisiol. Chem., 363, p. 1043, 1982). Native prourokinase (proUK) is a single chain (54000 daltons) form of urokinase and is also termed single chain urokinase type plasminogen activator (scu-PA). As stated before, proUK displays a fibrin-specific thrombolytic activity and is therefore a better thrombolytic agent compared to the presently used high or low molecular weight urokinases.
In order to produce prourokinase, the authors of the present invention have developed a recombinant DNA procedure which allows the preparation of large amounts of the proUK polypeptidic chain.
Several methods have been described in the scientific and patent literature for the production of proUK (Holmes, W. E., Pennica, D., Blaber, M., Rey, M. W., Gunzler, W. A., Steffens, G. J. and Heynecker, H. L.; Biotechnology, 3 , p. 923, 1985; European Patent Application 0092182). However, the method described within the text of the present invention exploits parameters known to be important for the expression of heterologous proteins in E. coli, but whose combination has never been applied before to the production of recombinant proUK.
The main parameters, whose combination contributes to the establishment of a recombinant strain of E. coli, able to produce proUK, and which represents the object of the present invention, are the E. coli promoter Ptrp, the Shine-Dalgarno sequence MS-2 from the phage MS-2, and E. coli strains of the type B as hosts for the expression of the human proUK gene (see below).
Such combination is crucial. Substitution of one of these parameters with an alternative expression signal may not yield as much proUK.
Accordingly, object of the present invention is a method for the preparation of non glycosilated pro-UK, characterized in that non-glycosilated pro-UK is expressed under the control of the E. coli promoter Ptrp and the Shine-Dalgarno sequence MS-2 by E. coli B.